The heat shock response (HSR) is a powerful transcriptional program which acts genome-wide, not only to restore the normal protein folding environment through the induction of heat shock proteins but as more recent work has shown to re-shape global cellular pathways controlling survival, growth and metabolism. In mammals, this response is regulated primarily by Heat Shock Factor 1 (HSF1), a transcription factor whose mode of action has been conserved in broad outline across all eukaryotes. Surprisingly, our recent work has shown that the many beneficial effects of HSF1 known to enhance the survival of organisms under stress come at the cost of facilitating the initiation and maintenance of cancers in mouse models and diverse human tumor lines driven by a variety of underlying oncogenic lesions. Acting at a global systems level, HSF1 function permits cells to survive the drastic imbalances in signaling and profound alterations in DNA, protein and energy metabolism that occur during malignant transformation. A novel therapeutic opportunity has been highlighted by our demonstration that knockdown of HSF1 expression using genetic techniques is well tolerated in normal cells and whole animals but that malignant cells display a profound dependence on this "non-oncogene." Relatively limited efforts have been directed so far at discovering inhibitors of this innovative target. The few HSF1 inhibitors that have been reported demonstrate limited specificity with prominent effects on general transcription and translation and poorly defined mechanisms of action. To address this deficiency and develop potent and selective inhibitors of HSF1 function, this project will join our extensive experience in the heat shock response, experimental therapeutics and cancer biology with the expertise in high-throughput screening and chemical probe optimization now available through the NIH Molecular Libraries Probe Production Center Network to accomplish the following specific aims: Aim1: Identify specific inhibitors of HSF1 using an optimized high throughput cell-based dual reporter assay that will markedly reduce false positives during the primary screening process. Aim 2: Evaluate the potency, specificity and mode of action of compounds using secondary and counter-screening assays in order to guide the selection of screen hits for analog synthesis and support their optimization into useful chemical biological probes. To validate our approach, we have completed a pilot screen with the Broad Institute Chemical Biology Platform using a collection of 70,000 known bioactive, small drug-like and purified natural products. As proof of principle, this screen identified a family of natural products that specifically inhibit the activation of HSF1. The discovery of additional inhibitors that target HSF1 function at mechanistically distinct regulatory steps in its activation will shed light on its multifaceted role in cancer biology and will provide important leads for pre- clinical studies in the chemoprevention and chemotherapy of a wide range of human cancers. PUBLIC HEALTH RELEVANCE: Heat Shock Factor 1 (HSF1) is a multifaceted and powerful regulator of tumorigenesis and malignant progression. While normal cells are not dependent upon HSF1, tumor cells are critically dependent upon it for survival. Since no specific and potent inhibitors of this innovative target exist, we propose to identify chemical probe inhibitors of HSF1 to better understand the role of HSF1 in cancer and to serve as tools in the pre- clinical development of chemopreventative and chemotherapeutic agents.